Interaction sites on phosphorylase kinase for calmodulin |
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Authors: | Heilmeyer Ludwig M G Gerschinski Andrea M Meyer Helmut E Jennissen Herbert P |
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Institution: | (1) Institut für Physiologische Chemie, Abteilung für Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, 44780 Bochum, Germany;(2) Institut für Physiologische Chemie, Universität-GHS-Essen, Universitätsklinikum, Hufelandstraße 55, 45147 Essen, Germany |
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Abstract: | Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg2+/ADP binding to by calmodulin is presented. |
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Keywords: | phosphorylase kinase calmodulin calmodulin-binding peptides Ca2+-binding |
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